The gonadotropin-releasing hormone receptor (GnRH-R) serves as a critical integrator of the reproductive system. GnRH and its analogs are extensively employed in the treatment of a wide spectrum of diseases including infertility, cryptorchidism, polycystic ovarian syndrome, leoiomyomata, and breast, ovarian and prostatic cancer. GnRH analogs are also being exploited as the basis of novel contraceptive systems in both men and women. The collaborating laboratories of Dr. S. Sealfon (PI on parent NIH proposal) and Dr. R. Millar (co-investigator on parent proposal) have been productively investigating the precise interaction of mammalian GnRH with its receptor at a molecular level. The current proposal for FIRCA support envisages a collaboration centered in Dr. Millar's laboratory that extends beyond the parent proposal to investigate the molecular and functional evolution of the GnRH-R in non-mammalian vertebrates and in protovertebrates. The structure of vertebrate GnRHs have been determined in species spanning over 500 million years of evolution. The specificities of the GnRH-R vary considerably among different vertebrates. These "experiments of nature" can provide considerable insight into the structures of these receptors and the sequence and structural motifs which recognize the conserved and distinct features of the various natural ligands. In addition, as more than one GnRH has been identified in a number of non-mammalian species, it is conceivable that the study of non-mammalian GnRH-Rs will lead to the identification of more than a single GnRH-R in a given species. In the first part of the investigation, the primary amino acid sequences of GnRH- Rs will be determined, at least in part, from fish, amphibian, reptile, bird and protochordate species by molecular cloning. Partial length sequences likely to represent the desired clones for most of these targets have already been isolated by PCR. Full length clones for at least two non-mammalian species will be isolated by PCR and conventional library screening techniques. The putative clones will be confirmed by expression in mammalian cells and by pharmacological characterization. Analysis of the primary sequences obtained will provide insight into the conserved residues likely to be involved in recognizing common features in the various ligands and into the distinct sequence elements likely to mediate ligand selectivity. The sequence differences will be investigated by site- directed mutagenesis and by the construction of receptor chimeras in order to elucidate the molecular basis of the differences in selectivity of the various receptors. These constructs will be probed by labeled agonist and antagonist binding and by monitoring agonist-stimulation and antagonist inhibition of phosphoinositol turnover. By identifying the receptor domains, motifs and individual residues that mediate ligand selectivity of non-mammalian GnRH-Rs the proposed studies will provide insight into the molecular evolution of GnRH-R selectivity and into the receptor domains that constitute the GnRH-R binding pocket.